There is a pressing need to develop a viable long-term storage method to safeguard the tens of thousands of genetic variants of Drosophila melanogaster that are used in genetics, developmental biology, molecular biology and human disease research. Previous attempts to cryopreserve Drosophila embryos using traditional techniques met with limited success and are not in use. In contrast to embryos, larvae of many insect species, including many dipterans (flies), are naturally able to survive freezing and chilling. The proposed research will therefore use larvae of D. melanogaster and related species as targets for cryopreservation. We will identify Drosophila species that tolerate freezing or chilling, and use these, in conjunction with the freeze tolerant drosophilid Chymomyza costata, as models to be used in developing a method to cryopreserve D. melanogaster. An important obstacle is the susceptibility of D. melanogaster to chilling (non-freezing) injury during cooling. It is essential to understand the nature of chilling injury in order to mitigate its effects. We will therefore investigate methods to improve the ability of D. melanogaster larvae to survive chilling, and to mitigate chilling damage that does occur. First, we will explore the plasticity of chill tolerance in response to changing conditions during acclimation, cold exposure and development. Second, we will investigate tissue-specific patterns of chilling injury, and their biochemical correlates in D. melanogaster. We will then compare the causes of chilling injury between D. melanogaster and chill-tolerant species to determine significant differences that may suggest a strategy for the mitigation of chilling injury. Finally, we will use infra-red and x-ray imaging to compare the freezing process in larvae of C. costata and D. melanogaster, and use non-genetic techniques to manipulate the freezing process in the latter. The ultimate goal is to cryopreserve D. melanogaster, an achievement that will greatly increase the utility of the Drosophila model for human disease research. [unreadable] [unreadable] [unreadable]